In vitro and in vivo nutrient transfer limits must be overcome in order to increase the feasibility of cell based therapeutic strategies. To enhance in vitro nutrient transport, the tubular perfusion system (TPS), a novel bioreactor recently developed by our laboratory, will dynamically culture human mesenchymal stem cells (hMSCs) in three dimensional scaffolds. This system utilizes an elegant design to create an effective cell culture environment without the drawbacks often associated with more complicated perfusion systems. The TPS design consists of hMSCs encapsulated in alginate beads which are tightly packed in a tubular growth chamber. Perfusing media through this growth chamber enhances nutrient transfer while exposing the cells to shear stress. To enhance in vivo vascularization, a prevascular network will be templated within the engineered tissue prior to implantation. To accomplish this, the TPS bioreactor will be optimized to support a coculture of endothelial cells and hMSCs. To examine this strategy of enhanced in vitro nutrient transport and in vivo vascularization, we propose first to investigate the TPS culture environment, particularly alginate bead size, bead composition, and media perfusion rate, that promotes hMSC proliferation and subsequent osteoblastic differentiation. Second, we propose to investigate the impact of endothelial cell coculture parameters, specifically coculture ratio, on the development of a prevascular network as well as the proliferation and differentiation of hMSCs. Third, we propose to implement a synthetic polymer sleeve system to support successful implantation of the in vitro cultured tissue. This strategy allows for the in vitro culture of functional engineered tissue, provides an elegant method for the in vivo implantation of the tissue, and fosters rapid integration of the implanted tissue into the host vasculature. Successful completion of these studies will demonstrate the feasibility of this fundamental technology for enhanced in vitro and in vivo nutrient transfer within cell based devices. PUBLIC HEALTH RELEVANCE: Bone injuries resulting from trauma, tumor removal, or disease are often inadequately healed by the body's natural mechanisms. Current treatments for bone injuries have limited success. Regenerative medicine approaches often suggest successful in vitro culture of stem cells outside and rapid in vivo vascularization of the implanted tissue. To this end, we aim to develop strategies for the culture of mesenchymal stem cells, with a prevascularization network, using a novel bioreactor system.